The long-term goal of this research is two-fold. One is to gain insights into the mechanisms that control fate choice in the early developing nervous system. The principal tool is ES cells differentiated in culture to produce a model of normal CNS development. The model is powerful because large numbers of cells are produced and because gone targeting and other genetic manipulations are feasible. Using the advantages of this model we will investigate how early neural precursor cells choose a fate defined by expression of the Olig2 gene. Cells from this pathway eventually differentiate into motor neurons and oligodendrocytes. Retinoic acid and sonic hedgehog interact to specify na'ive cells to follow this fate. We will learn how these 2 pathways interact. Specified cells are capable of cell division. We have discovered how to induce dividing cells to undergo long-term division. Even though they divide continuously the cells keep their identity as members of the Olig2 lineage. Our working hypothesis is that normal early neural stem cells are indeed capable of sustained division. If true, this will provide large numbers of specified neural stem cells for many applications. The final stage of stem cell life is differentiation. We will investigate signals that are responsible for differentiation of Olig2 pathway cells. We have already discovered how to efficiently differentiate these cells into astrocytes and oligodendrocytes and will extend this work to neurons. The second long-term goal is to harness the potential that ES cells have for neural transplantation. While the potential is real, practical application demands major advances in basic understanding. It is theoretically possible to direct ES cells to coordinately differentiate into any single type of neural cell. Achievement of this goal even for a few cell lineages would open up new opportunities in transplantation research. Results of this project will provide proof-of-principle for this idea. A combination of instructive and selective strategies is being applied to the goal of getting pure populations. Cells from each stage of the Olig2 lineage will be available in large numbers and at high purity. Lessons learned from this lineage will be applicable to others. One strong possibility is that genetically engineered cells will be most efficacious in transplantation. The ES cell-based system is ideally suited to providing genetically engineered cells.